A dipeptyl sweetener, L-aspartyl-L-phenylalanine methyl ester, also known as aspartame, has been developed and commercialized. The demand of L-aspartic acid for use in the production of the sweetener has been increasing dramatically all over the world in recent years.
Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) is an enzyme which catalyzes the reversible conversion of fumaric acid and ammonia to L-aspartic acid, as well as the reverse reaction of deamination of L-aspartic acid: ##STR1## Yumoto et al., Physiol. Chem. Phys. 14:391-397 (1982) reported that the activity of aspartase from E. coli was enhanced 1.4-3.8 fold by limited proteolysis. The enhancement of activity was resulted from the cleavage of a peptide bond at certain region centered at arginine from the carboxy-terminal.
Murase et al., Biochem. Biophys. Res. Commun. 177:414-419 (1991) disclosed a mutant aspartase wherein the cysteine residue at position 430 in the amino acid sequence of the wild-type enzyme was substituted with tryptophan by site-directed mutagenesis. The catalytic activity of the mutant was 134% of that of the wild-type enzyme tinder standard assay conditions.
Zhang et al., Biochem. Biophys. Res. Commun. 192:15-21 (1993) described a mutant aspartase with 3.7-fold increase in catalytic activity than that of the wild-type enzyme. The mutant was prepared by site-directed mutagenesis to have an arginine residue at 126 position instead of lysine occurring, in the wild-type enzyme.
All these references are hereby incorporated by reference in their entirety and specific pertinent parts.
Despite of the above, there is a continuing need for new mutant aspartases with enhanced catalytic activity useful for efficiently producing L-aspartic acid.